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The voltage-dependent gate in MthK potassium channels is located at the selectivity filter

Nature Structural & Molecular Biology volume 20, pages 159–166 (2013)Cite this article

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Abstract

Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. Blockers bind in an aqueous cavity between two putative gates: an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location—an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.

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Figure 1: Block mechanism with two gate locations.
Figure 2: Block of open MthK.
Figure 3: Voltage dependence of open-state block and apparent affinities during inactivation.
Figure 4: Inactivation does not gate TBA access to the binding site.
Figure 5: bTBA and bbTBA block are consistent with state-independent access.
Figure 6: Summary of state-dependent MthK block.
Figure 7: TBSb difference density in the cavity supports blocker binding immediately below the selectivity filter.

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References

  1. Hille, B. Ion Channels of Excitable Membranes (Sinauer, Sunderland, Massachusetts, USA, 2001).

  2. Armstrong, C.M. & Binstock, L. Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. Gen. Physiol. 48, 859–872 (1965).

    Article  CAS  Google Scholar 

  3. Armstrong, C.M. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J. Gen. Physiol. 58, 413–437 (1971).

    Article  CAS  Google Scholar 

  4. Choi, K.L., Mossman, C., Aube, J. & Yellen, G. The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron 10, 533–541 (1993).

    Article  CAS  Google Scholar 

  5. Liu, Y., Holmgren, M., Jurman, M.E. & Yellen, G. Gated access to the pore of a voltage-dependent K+ channel. Neuron 19, 175–184 (1997).

    Article  Google Scholar 

  6. Holmgren, M., Smith, P.L. & Yellen, G. Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating. J. Gen. Physiol. 109, 527–535 (1997).

    Article  CAS  Google Scholar 

  7. Doyle, D.A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).

    Article  CAS  Google Scholar 

  8. Zhou, M., Morais-Cabral, J.H., Mann, S. & MacKinnon, R. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature 411, 657–661 (2001).

    Article  CAS  Google Scholar 

  9. Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002).

    Article  CAS  Google Scholar 

  10. Jiang, Y. et al. The open pore conformation of potassium channels. Nature 417, 523–526 (2002).

    Article  CAS  Google Scholar 

  11. Yellen, G. The moving parts of voltage-gated ion channels. Q. Rev. Biophys. 31, 239–295 (1998).

    Article  CAS  Google Scholar 

  12. Hoshi, T., Zagotta, W.N. & Aldrich, R.W. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250, 533–538 (1990).

    Article  CAS  Google Scholar 

  13. Kurata, H.T. & Fedida, D. A structural interpretation of voltage-gated potassium channel inactivation. Prog. Biophys. Mol. Biol. 92, 185–208 (2006).

    Article  CAS  Google Scholar 

  14. Choi, K.L., Aldrich, R.W. & Yellen, G. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc. Natl. Acad. Sci. USA 88, 5092–5095 (1991).

    Article  CAS  Google Scholar 

  15. McCoy, J.G. & Nimigean, C.M. Structural correlates of selectivity and inactivation in potassium channels. Biochim. Biophys. Acta 1818, 272–285 (2012).

    Article  CAS  Google Scholar 

  16. Cuello, L.G., Jogini, V., Cortes, D.M. & Perozo, E. Structural mechanism of C-type inactivation in K+ channels. Nature 466, 203–208 (2010).

    Article  CAS  Google Scholar 

  17. Kurata, H.T., Wang, Z. & Fedida, D. NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels. J. Gen. Physiol. 123, 505–520 (2004).

    Article  CAS  Google Scholar 

  18. Baukrowitz, T. & Yellen, G. Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels. Proc. Natl. Acad. Sci. USA 93, 13357–13361 (1996).

    Article  CAS  Google Scholar 

  19. Panyi, G. & Deutsch, C. Cross talk between activation and slow inactivation gates of Shaker potassium channels. J. Gen. Physiol. 128, 547–559 (2006).

    Article  CAS  Google Scholar 

  20. Panyi, G. & Deutsch, C. Probing the cavity of the slow inactivated conformation of Shaker potassium channels. J. Gen. Physiol. 129, 403–418 (2007).

    Article  CAS  Google Scholar 

  21. Flynn, G.E. & Zagotta, W.N. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron 30, 689–698 (2001).

    Article  CAS  Google Scholar 

  22. Bruening-Wright, A., Lee, W.S., Adelman, J.P. & Maylie, J. Evidence for a deep pore activation gate in small conductance Ca2+-activated K+ channels. J. Gen. Physiol. 130, 601–610 (2007).

    Article  Google Scholar 

  23. Proks, P., Antcliff, J.F. & Ashcroft, F.M. The ligand-sensitive gate of a potassium channel lies close to the selectivity filter. EMBO Rep. 4, 70–75 (2003).

    Article  CAS  Google Scholar 

  24. Contreras, J.E., Srikumar, D. & Holmgren, M. Gating at the selectivity filter in cyclic nucleotide-gated channels. Proc. Natl. Acad. Sci. USA 105, 3310–3314 (2008).

    Article  CAS  Google Scholar 

  25. Klein, H. et al. Structural determinants of the closed KCa3.1 channel pore in relation to channel gating: results from a substituted cysteine accessibility analysis. J. Gen. Physiol. 129, 299–315 (2007).

    Article  CAS  Google Scholar 

  26. Wilkens, C.M. & Aldrich, R.W. State-independent block of BK channels by an intracellular quaternary ammonium. J. Gen. Physiol. 128, 347–364 (2006).

    Article  CAS  Google Scholar 

  27. Thompson, J. & Begenisich, T. Selectivity filter gating in large-conductance Ca2+-activated K+ channels. J. Gen. Physiol. 139, 235–244 (2012).

    Article  CAS  Google Scholar 

  28. Tang, Q.Y., Zeng, X.H. & Lingle, C.J. Closed-channel block of BK potassium channels by bbTBA requires partial activation. J. Gen. Physiol. 134, 409–436 (2009).

    Article  CAS  Google Scholar 

  29. Zhou, Y., Xia, X.M. & Lingle, C.J. Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region. Proc. Natl. Acad. Sci. USA 108, 12161–12166 (2011).

    Article  CAS  Google Scholar 

  30. Cui, J., Yang, H. & Lee, U.S. Molecular mechanisms of BK channel activation. Cell. Mol. Life Sci. 66, 852–875 (2009).

    Article  CAS  Google Scholar 

  31. Yuan, P., Leonetti, M.D., Hsiung, Y. & MacKinnon, R. Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. Nature 481, 94–97 (2012).

    Article  CAS  Google Scholar 

  32. Wu, Y., Yang, Y., Ye, S. & Jiang, Y. Structure of the gating ring from the human large-conductance Ca2+-gated K+ channel. Nature 466, 393–397 (2010).

    Article  CAS  Google Scholar 

  33. Zadek, B. & Nimigean, C.M. Calcium-dependent gating of MthK, a prokaryotic potassium channel. J. Gen. Physiol. 127, 673–685 (2006).

    Article  CAS  Google Scholar 

  34. Ye, S., Li, Y. & Jiang, Y. Novel insights into K+ selectivity from high-resolution structures of an open K+ channel pore. Nat. Struct. Mol. Biol. 17, 1019–1023 (2010).

    Article  CAS  Google Scholar 

  35. Thomson, A.S. & Rothberg, B.S. Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel. J. Gen. Physiol. 136, 569–579 (2010).

    Article  CAS  Google Scholar 

  36. Martínez-François, J.R. & Lu, Z. Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore. J. Gen. Physiol. 135, 149–167 (2010).

    Article  Google Scholar 

  37. Lenaeus, M.J., Vamvouka, M., Focia, P.J. & Gross, A. Structural basis of TEA blockade in a model potassium channel. Nat. Struct. Mol. Biol. 12, 454–459 (2005).

    Article  CAS  Google Scholar 

  38. Yohannan, S., Hu, Y. & Zhou, Y. Crystallographic study of the tetrabutylammonium block to the KcsA K+ channel. J. Mol. Biol. 366, 806–814 (2007).

    Article  CAS  Google Scholar 

  39. Faraldo-Gómez, J.D. et al. Mechanism of intracellular block of the KcsA K+ channel by tetrabutylammonium: insights from X-ray crystallography, electrophysiology and replica-exchange molecular dynamics simulations. J. Mol. Biol. 365, 649–662 (2007).

    Article  Google Scholar 

  40. French, R.J. & Shoukimas, J.J. Blockage of squid axon potassium conductance by internal tetra-N-alkylammonium ions of various sizes. Biophys. J. 34, 271–291 (1981).

    Article  CAS  Google Scholar 

  41. Guo, D. & Lu, Z. Kinetics of inward-rectifier K+ channel block by quaternary alkylammonium ions. Dimension and properties of the inner pore. J. Gen. Physiol. 117, 395–406 (2001).

    Article  CAS  Google Scholar 

  42. Spassova, M. & Lu, Z. Coupled ion movement underlies rectification in an inward-rectifier K+ channel. J. Gen. Physiol. 112, 211–221 (1998).

    Article  CAS  Google Scholar 

  43. Pau, V.P., Abarca-Heidemann, K. & Rothberg, B.S. Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel. J. Gen. Physiol. 135, 509–526 (2010).

    Article  CAS  Google Scholar 

  44. Woodhull, A.M. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687–708 (1973).

    Article  CAS  Google Scholar 

  45. Thompson, J. & Begenisich, T. External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter. J. Gen. Physiol. 122, 239–246 (2003).

    Article  CAS  Google Scholar 

  46. Heginbotham, L. & Kutluay, E. Revisiting voltage-dependent relief of block in ion channels: a mechanism independent of punchthrough. Biophys. J. 86, 3663–3670 (2004).

    Article  CAS  Google Scholar 

  47. Nimigean, C.M. & Miller, C. Na+ block and permeation in a K+ channel of known structure. J. Gen. Physiol. 120, 323–335 (2002).

    Article  CAS  Google Scholar 

  48. Rothberg, B.S., Shin, K.S., Phale, P.S. & Yellen, G. Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. J. Gen. Physiol. 119, 83–91 (2002).

    Article  CAS  Google Scholar 

  49. Contreras, J.E. & Holmgren, M. Access of quaternary ammonium blockers to the internal pore of cyclic nucleotide-gated channels: implications for the location of the gate. J. Gen. Physiol. 127, 481–494 (2006).

    Article  CAS  Google Scholar 

  50. Contreras, J.E. et al. Voltage profile along the permeation pathway of an open channel. Biophys. J. 99, 2863–2869 (2010).

    Article  CAS  Google Scholar 

  51. Jogini, V. & Roux, B. Electrostatics of the intracellular vestibule of K+ channels. J. Mol. Biol. 354, 272–288 (2005).

    Article  CAS  Google Scholar 

  52. Geng, Y., Niu, X. & Magleby, K.L. Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume. J. Gen. Physiol. 137, 533–548 (2011).

    Article  CAS  Google Scholar 

  53. Chen, X. & Aldrich, R.W. Charge substitution for a deep-pore residue reveals structural dynamics during BK channel gating. J. Gen. Physiol. 138, 137–154 (2011).

    Article  CAS  Google Scholar 

  54. Li, W. & Aldrich, R.W. State-dependent block of BK channels by synthesized shaker ball peptides. J. Gen. Physiol. 128, 423–441 (2006).

    Article  CAS  Google Scholar 

  55. Wang, D.T., Hill, A.P., Mann, S.A., Tan, P.S. & Vandenberg, J.I. Mapping the sequence of conformational changes underlying selectivity filter gating in the Kv11.1 potassium channel. Nat. Struct. Mol. Biol. 18, 35–41 (2011).

    Article  Google Scholar 

  56. Cuello, L.G. et al. Structural basis for the coupling between activation and inactivation gates in K+ channels. Nature 466, 272–275 (2010).

    Article  CAS  Google Scholar 

  57. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  58. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  59. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  Google Scholar 

  60. Qin, F., Auerbach, A. & Sachs, F. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys. J. 70, 264–280 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Y. Jiang for advice on MthK crystal preparation and J. Rubin for technical assistance. B. Rothberg (Temple University, Philadelphia, Pennsylvania) provided the MthK-pQE82 construct. We also thank the staff of the X25 beamline at the National Synchrotron Light Source, Brookhaven National Laboratory, for their help. This work was supported by an NRSA postdoctoral fellowship from the US National Institutes of Health (F32GM087865) to D.J.P. and a National Institutes of Health grant (RO1GM088352) to C.M.N.

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Authors and Affiliations

  1. Department of Anesthesiology, Weill Cornell Medical College, New York, New York, USA

    David J Posson, Jason G McCoy & Crina M Nimigean

  2. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA

    Crina M Nimigean

  3. Department of Biochemistry, Weill Cornell Medical College, New York, New York, USA

    Crina M Nimigean

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  1. David J Posson
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  2. Jason G McCoy
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  3. Crina M Nimigean
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Contributions

D.J.P. and C.M.N. designed the research; D.J.P. acquired and analyzed single-channel data; D.J.P. and J.G.M. acquired and analyzed crystallographic data; D.J.P., J.G.M. and C.M.N. wrote the paper.

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Correspondence to David J Posson or Crina M Nimigean.

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Posson, D., McCoy, J. & Nimigean, C. The voltage-dependent gate in MthK potassium channels is located at the selectivity filter. Nat Struct Mol Biol 20, 159–166 (2013). https://doi.org/10.1038/nsmb.2473

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